小G蛋白Rab5对表达在HEK293细胞上大电导钙激活钾通道的影响

发布时间：2018-06-10 03:22

本文选题：Rab + 大电导钙激活钾通道　；参考：《西南医科大学》2017年硕士论文

【摘要】：目的:众所周知,大电导钙激活钾通道(BK_(Ca))可以将人体不同组织细胞内的钙离子信号和细胞兴奋性相联系,这种生理功能在调节人体的血液流动、尿液排出、免疫应答等多种生命活动中具有极其重要的作用。BK_(Ca)通道是人体血管平滑肌细胞膜上的主要离子通道,在血管舒缩功能调控中起着至关重要的作用。BK_(Ca)通道由4个a亚基与具有调节功能的b亚基组成,其中b亚单位是BK_(Ca)通道在人体血管平滑肌细胞上最为重要的辅助亚单位,是通道蛋白执行其生理功能的关键性因素之一,如果b亚单位出现生理功能异常,就可能引起多种心血管疾病(如高血压等)。BK_(Ca)通道蛋白在内质网合成、高尔基体加工之后,还需经过一系列复杂的转运机制并组装到质膜上,才能发挥其生理功能。小G蛋白家族成员中的Rab在质膜内侧及多种胞内囊泡结构中存在,具有调节细胞信号转导、囊泡转运和离子通道活动等生理功能。结合既往的研究,提示Rab和BK_(Ca)通道蛋白可能存在某种相互联系,但是全面深入的研究甚少,为了更好的研究BK_(Ca)通道蛋白的调节机制,我们选取了Rab5作为研究对象,展开实验,探讨小G蛋白Rab5对HEK293细胞大电导钙激活钾(BK_(Ca))通道的影响。方法:根据以往的研究经验,我们选用的实验细胞为人胚肾293细胞系。首先将含有人血管平滑肌BKca通道a亚单位(hSlo1)的Flag和GFP双标记表达质粒(Flag-hSlo1-GFP)使用脂质体转染法和不同形式的Rab5表达质粒(野生型WT,显性负向型DN,持续激活型CA)转染至培养的HEK293细胞,使用膜片钳技术、Western blotting和流式细胞术,研究小G蛋白Rab5对BK_(Ca)通道的细胞宏观电流、BK_(Ca)通道的膜蛋白在HEK293细胞膜上表达水平和BK_(Ca)通道的转运蛋白的影响。结果:实验结果表明,表达在HEK293细胞上的大电导钙激活钾(BK_(Ca))通道和天然BK_(Ca)通道几乎具有相似的电生理学特性,膜片钳实验表明在全细胞构型下,当细胞膜电位(V_m)达到~+60mV时,Rab5 WT和Rab5 CA可以分别增加BKca通道宏观电流密度由49.19±3.76pA/pF增加到68.38±5.33pA/pF(P0.05,n=6)和87.16±3.79pA/pF(P0.05,n=6)。而Rab5DN减少BKca通道宏观电流密度,由49.19±3.76pA/pF减少到34.68±3.37pA/pF(P0.05,n=6)。Western blotting实验结果显示,Rab5 WT和Rab5 CA增加BK_(Ca)通道蛋白在HEK293细胞膜上的表达水平,相对表达量分别是对照组的1.24±0.08倍(Rab5 WT)和1.42±0.13倍(Rab5CA)(P0.05),而Rab5 DN减少BK_(Ca)通道蛋白在HEK293细胞膜上的表达水平,相对表达量是对照组的0.73±0.11(P0.05),两者都具有显著的统计学意义。流式细胞术实验结果显示Rab5 WT和Rab5 CA可以明显增加Flag~+/GFP~+比值,而Rab5 DN明显减小Flag~+/GFP~+比值(P0.05或0.01),这表明Rab5 WT和Rab5 CA明显促进了BK_(Ca)通道蛋白向细胞膜上的转运。结论:Rab5能够明显增加表达在HEK293细胞上的BK_(Ca)电流及细胞膜上的表达,并可能促进了BK_(Ca)通道向细胞膜的转运过程。
[Abstract]:Objective: it is well known that large conductance calcium-activated potassium channel (BK) can link calcium signals in different tissues and cells to cell excitability, a physiological function that regulates the flow of blood and excretion of urine. The immune response plays an extremely important role in many life activities. BK\ + Ca) channel is the main ion channel in human vascular smooth muscle cell membrane. In the regulation of vasomotor and contraction function, the BKG Ca) channel is composed of four subunits a and b subunits with regulatory function, among which b subunit is the most important auxiliary subunit of BK subunit in human vascular smooth muscle cells. It is one of the key factors for channel protein to perform its physiological function. If the subunit b has abnormal physiological function, it may cause the synthesis of channel proteins in the endoplasmic reticulum (ER) and the processing of Golgi body in various cardiovascular diseases (such as hypertension, etc.). It also needs a series of complex transport mechanisms and assembly to the plasma membrane to play its physiological function. The Rab of small G protein family exists in the medial membrane of plasma membrane and many kinds of intracellular vesicle structures, and has the physiological function of regulating cell signal transduction, vesicle transport and ion channel activity. In combination with previous studies, it is suggested that there may be some interrelation between Rab and BKS Ca-channel proteins, but there are few comprehensive and in-depth studies. In order to better study the regulation mechanism of BKKCa-Ca-channel proteins, we selected Rab5 as the object of study and carried out experiments. To investigate the effect of small G protein Rab5 on the large conductance calcium activated potassium (BK / C) channel in HEK293 cells. Methods: according to the previous research experience, we selected the human embryonic kidney 293 cell line. Flag and GFP double labeled expression plasmids, Flag-hSlo1-GFP1, containing a subunit of human vascular smooth muscle BKCA channel, were first transfected into HEK293 cells by liposome transfection and different forms of Rab5 expression plasmids (wild type WT, dominant negative type DNs, continuous activated CA1). Using patch clamp technique, Western blotting and flow cytometry were used to study the effect of small G protein Rab5 on the expression of membrane protein on the membrane of HEK293 cell membrane and the transport protein of BK-1 Ca2 channel. Results: the experimental results showed that the large conductance calcium-activated potassium (BK) and the natural BK-1 / CaC) channels expressed in HEK293 cells had almost similar electrophysiological characteristics, and patch clamp experiments showed that in the whole cell configuration, the two channels had similar electrophysiological characteristics. Rab5WT and Rab5CA could increase the macroscopic current density of BKCA channel from 49.19 卤3.76pA / PF to 68.38 卤5.33pA / P0.05P0.05N ~ (6) and 87.16 卤3.79pAp / p ~ (0.05p ~ (6) respectively when the cell membrane potential reached ~ 60mV. However, Rab5DN decreased the macroscopic current density of BKca channel from 49.19 卤3.76pA / PF to 34.68 卤3.37pFN / P0.05P0.05P0.05P0.05P0.05P0. Western blotting results showed that Rab5WT and Rab5CA increased the expression level of BKSI-CaA channel protein on HEK293 cell membrane. The relative expression was 1.24 卤0.08 times higher than that of control group (1.24 卤0.08 times) and 1.42 卤0.13 times of Rab5CAA (P 0.05), respectively, while Rab5 DN reduced the expression level of BKapphica-channel protein on HEK293 cell membrane. The relative expression level of Rab5DN was 0.73 卤0.11P0.05in the control group. Both of them had significant statistical significance. The results of flow cytometry showed that Rab5WT and Rab5CA could significantly increase the Flag- / GFP~ ratio, while Rab5DN significantly decreased the Flag- / GFP~ ratio (P0.05 or 0.01), which indicated that Rab5WT and Rab5CA significantly promoted the transport of BKGFP-) channel protein to the cell membrane. ConclusionWowRab5 can obviously increase the BKS Ca) current expressed in HEK293 cells and the expression on the cell membrane, and may promote the transport process of BKS Ca) channel to the cell membrane.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R33

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